PROJECT SUMMARY/ABSTRACT Autologous vein grafts are the most common and most effective bypass grafts when used in the heart to treat coronary artery disease or in the lower extremity to prevent amputations. Nonetheless the delayed failure rate of vein grafts is about 30%, almost all due to intimal hyperpalsia (IH). IH, in turn, is an integral part of the response to implantation injury when the vein is moved from the low pressure, vasa vasorum dependent physiology, to the high pressure, luminal dependent physiology of the arterial system. Our goal is to minimize this implantation injury, and thereby diminish the maladaptive, pathologic remodeling associated with IH. Our experimental design has always been guided by practical considerations whereby the therapeutic intervention could be conducted within the clinical constraints of the operating room. Our strategy has been to first determine the time dependent gene expression response (transcriptome), 1 day to 30 days after implantation, followed by identifying the most important ?Hub? genes that drive the pathologic remodeling. Our therapeutic approach is to silence these pathogenic hub genes in the vascular wall. Therefore, we knocked down and tested two of our most promising targets, Thrombospondin-2 (TSP-2) and Myristoylated alanine-rich C-kinase substrate (MARCKS) in two different reproducible large animal models to document molecular and histologic outcomes. Going forward we are going to supplement our therapeutic regimen by overexpressing the atheroprotective, anti-inflammatory gene, TNF-alpha induced protein-3 (TNFAIP3 or A20), using clinically applicable vasculotropic gene therapy vectors. A multimodal and bidirectional (knockdown of atherogenic and overexpression of atheroprotective genes) therapeutic regimen may well be required to address the complex pathogenesis of IH. Using, for the first time, the state-of-the-art integrated single cell (Sc), coupled with bulk RNA-sequencing we will map the transcriptomic granularity of the VG heterogeneous response to implantation injury and to therapy. This could determine more precisely the role of vascular and non-vascular, yet influential, cellular subsets such as immune cells, myofibroblasts etc. in the pathogenesis of VG implantation injury. Our preliminary results in a canine vein graft model with high biofidelity to human disease uncovers pertinent novel information, highlighting a so far underestimated contribution of pathogenic T helper 1 cells (Th1) to VG remodeling. These new experiments will bring us to preclinical readiness in a strategy designed to use the most advanced delivery systems and molecular technologies to reduce implantation injury to vein grafts and diminish IH in other vascular diseases. Our research team is structured to take advantage of the MPI format to coalesce the broad expertise required to bring this project to fruition and maintain an enduring focus on the IH problem.